Tech Note: Simplified protocol for SMARTer Pico kit

Abstract

The SMARTer(R) Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian kit from Takara(R) (SMARTer Pico) has proved successful and reliable in generating stranded RNA Illumina libraries from degraded total RNA and ultra-low input amounts of total RNA below detection level. Here we attempted to streamline and simplify the library prep protocol at the key fragmentation step bottleneck. Our key findings were that reduced fragmentation times neither affect the depletion efficiency, nor the library complexity. Skipping the fragmentation resulted in longer libraries when examined in the capillary electrophoresis but this was compensated for during sequencing, as long fragments are less likely to form clusters during sequencing. Skipping the fragmentation also affected the gene body coverage, where a bias to the 5 end was observed though this compromised neither the data quality, complexity nor reproducibility. Additionally, using 16 PCR cycles seems to have little effect on the library complexity. Overall, we can see that sample input is the key to library complexity and reproducibility, while fragmentation time has less impact on data. Is total RNA fragmentation needed? Is rRNA depletion affected by fragmentation time? Or affected by input? Can we get reliable data when starting with low input?